The goal of this project is to identify novel targets and drugs for the treatment and prophylaxis of smallpox. Anti-poxvirus drug targets are a pressing issue, given the concern that smallpox may be used as a bioterrorism weapon against an unvaccinated population. We propose to discover new inhibitors of poxvirus replication targeted to essential virus-encoded enzymes that are required for viral gene expression and DNA metabolism. The poxvirus mRNA capping apparatus, consisting of RNA triphosphatase. RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase enzymes, is a promising drug target because the organization of the three catalytic sites is distinct from that of human host cell capping system. The poxvirus type 18 DNA topoisomerase is an attractive target in light of its unique DNA recognition specificity, compact structure, and distinctive pharmacological sensitivities compared to human topoisomerase I. The specific aims of this application are: (1) To identify small molecules that bind to the target viral enzymes by in vitro screening of an encoded split-synthesis combinatorial library immobilized on a solid bead support (one compound per bead). (2) To test the individual compounds identified in the primary screen for their ability to inhibit the catalytic activities of the target triphosphatase, guanylyltransferase, methyltransferase, and topoisomerase enzymes. (3) To dissect the mechanisms of inhibition of catalytic activity by the compounds identified in the secondary screen, via kinetic analysis of the component steps of the capping and topoisomerase reactions. (4) To assay the enzyme inhibitors for their effects on vaccinia virus replication in cell culture, using plaque reduction and one-step growth assay methods. (5) To evaluate the mechanism of antiviral action by assessing the effects of the lead drug compounds on the major landmarks of the poxvirus replication cycle: viral mRNA and protein synthesis, DNA replication, telomere resolution, and virion morphogenesis.